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Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. dimension and properties of the inner pore.

Guo D, Lu Z - J. Gen. Physiol. (2001)

Bottom Line: This remarkable difference in QA selectivity between the two channels results primarily from differing QA unbinding kinetics.The apparent rate constant for binding (kon) of all examined QAs is significantly smaller than expected for a diffusion-limited process.These observations argue that between the intracellular solution and the QA-binding locus, there exists a constricted pathway, whose dimension ( approximately 9 A) is comparable to that of a K+ ion with a single H2O shell.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
We examined block of two inward-rectifier K+ channels, IRK1 and ROMK1, by a series of intracellular symmetric quaternary alkylammonium ions (QAs) whose side chains contain one to five methylene groups. As shown previously, the ROMK1 channels bind larger QAs with higher affinity. In contrast, the IRK1 channels strongly select TEA over smaller or larger QAs. This remarkable difference in QA selectivity between the two channels results primarily from differing QA unbinding kinetics. The apparent rate constant for binding (kon) of all examined QAs is significantly smaller than expected for a diffusion-limited process. Furthermore, a large ( approximately 30-fold) drop in kon occurs when the number of methylene groups in QAs increases from three to four. These observations argue that between the intracellular solution and the QA-binding locus, there exists a constricted pathway, whose dimension ( approximately 9 A) is comparable to that of a K+ ion with a single H2O shell.

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Analysis of the voltage jump–induced IRK1 current relaxations in the presence of QAs. (A) For a given QA, the reciprocals of the time constants (1/τ; mean ± SEM, n = 5) obtained from the fits as shown in Fig. 3 are plotted against concentration and fitted with straight lines. (B and C) The natural logarithms of kon and koff (mean ± SEM, n = 5) are plotted against membrane voltage, respectively. The lines through the data are fits of the equation: ln k = ln k(0 mV) ± zFV/RT. The values of k(0 mV) and z thus obtained are summarized in Fig. 10.
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Figure 4: Analysis of the voltage jump–induced IRK1 current relaxations in the presence of QAs. (A) For a given QA, the reciprocals of the time constants (1/τ; mean ± SEM, n = 5) obtained from the fits as shown in Fig. 3 are plotted against concentration and fitted with straight lines. (B and C) The natural logarithms of kon and koff (mean ± SEM, n = 5) are plotted against membrane voltage, respectively. The lines through the data are fits of the equation: ln k = ln k(0 mV) ± zFV/RT. The values of k(0 mV) and z thus obtained are summarized in Fig. 10.

Mentions: Fig. 3 A shows the outward current transients induced by stepping membrane voltage from the −100-mV prepulse to the various test voltages indicated, in the presence of a fixed concentration of one of the four QAs (the kinetics of current transients in the presence of TMA are too fast to be resolved by our recording system; Fig. 1). Fig. 3 B shows current transients at several concentrations of one of the fours QAs but at a fixed test voltage. The smooth curves superimposed on the current records in both A and B are single-exponential fits. The reciprocal of the single-exponential time constant at a given test voltage is plotted against QA concentration in Fig. 4 A. Assuming that one QA molecule blocks one channel, we determined the apparent “on” rate constant (kon) for the tested QA from the slope of the linear fit in Fig. 4 A and the “off” rate constant (koff) from the product of kon and the corresponding Kd. The natural logarithms of kon and koff are plotted against membrane voltage in Fig. 4 (B and C, respectively). The lines superimposed on the data in Fig. 4 (B and C) are fits of an equation, ln k = ln k(0 mV) ± zFV/RT. From the fits, we determined kon and koff at 0 mV and the corresponding valence factors (zon and zoff). Since the values of zon and zoff for a given QA are comparable, the voltage dependence of the dissociation constant reflects a similar influence of membrane voltage on both kon and koff.


Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. dimension and properties of the inner pore.

Guo D, Lu Z - J. Gen. Physiol. (2001)

Analysis of the voltage jump–induced IRK1 current relaxations in the presence of QAs. (A) For a given QA, the reciprocals of the time constants (1/τ; mean ± SEM, n = 5) obtained from the fits as shown in Fig. 3 are plotted against concentration and fitted with straight lines. (B and C) The natural logarithms of kon and koff (mean ± SEM, n = 5) are plotted against membrane voltage, respectively. The lines through the data are fits of the equation: ln k = ln k(0 mV) ± zFV/RT. The values of k(0 mV) and z thus obtained are summarized in Fig. 10.
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Related In: Results  -  Collection

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Figure 4: Analysis of the voltage jump–induced IRK1 current relaxations in the presence of QAs. (A) For a given QA, the reciprocals of the time constants (1/τ; mean ± SEM, n = 5) obtained from the fits as shown in Fig. 3 are plotted against concentration and fitted with straight lines. (B and C) The natural logarithms of kon and koff (mean ± SEM, n = 5) are plotted against membrane voltage, respectively. The lines through the data are fits of the equation: ln k = ln k(0 mV) ± zFV/RT. The values of k(0 mV) and z thus obtained are summarized in Fig. 10.
Mentions: Fig. 3 A shows the outward current transients induced by stepping membrane voltage from the −100-mV prepulse to the various test voltages indicated, in the presence of a fixed concentration of one of the four QAs (the kinetics of current transients in the presence of TMA are too fast to be resolved by our recording system; Fig. 1). Fig. 3 B shows current transients at several concentrations of one of the fours QAs but at a fixed test voltage. The smooth curves superimposed on the current records in both A and B are single-exponential fits. The reciprocal of the single-exponential time constant at a given test voltage is plotted against QA concentration in Fig. 4 A. Assuming that one QA molecule blocks one channel, we determined the apparent “on” rate constant (kon) for the tested QA from the slope of the linear fit in Fig. 4 A and the “off” rate constant (koff) from the product of kon and the corresponding Kd. The natural logarithms of kon and koff are plotted against membrane voltage in Fig. 4 (B and C, respectively). The lines superimposed on the data in Fig. 4 (B and C) are fits of an equation, ln k = ln k(0 mV) ± zFV/RT. From the fits, we determined kon and koff at 0 mV and the corresponding valence factors (zon and zoff). Since the values of zon and zoff for a given QA are comparable, the voltage dependence of the dissociation constant reflects a similar influence of membrane voltage on both kon and koff.

Bottom Line: This remarkable difference in QA selectivity between the two channels results primarily from differing QA unbinding kinetics.The apparent rate constant for binding (kon) of all examined QAs is significantly smaller than expected for a diffusion-limited process.These observations argue that between the intracellular solution and the QA-binding locus, there exists a constricted pathway, whose dimension ( approximately 9 A) is comparable to that of a K+ ion with a single H2O shell.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
We examined block of two inward-rectifier K+ channels, IRK1 and ROMK1, by a series of intracellular symmetric quaternary alkylammonium ions (QAs) whose side chains contain one to five methylene groups. As shown previously, the ROMK1 channels bind larger QAs with higher affinity. In contrast, the IRK1 channels strongly select TEA over smaller or larger QAs. This remarkable difference in QA selectivity between the two channels results primarily from differing QA unbinding kinetics. The apparent rate constant for binding (kon) of all examined QAs is significantly smaller than expected for a diffusion-limited process. Furthermore, a large ( approximately 30-fold) drop in kon occurs when the number of methylene groups in QAs increases from three to four. These observations argue that between the intracellular solution and the QA-binding locus, there exists a constricted pathway, whose dimension ( approximately 9 A) is comparable to that of a K+ ion with a single H2O shell.

Show MeSH
Related in: MedlinePlus